Genomic surveillance: a potential shortcut for effective Chagas disease management.

Chagas disease is an enduring public health issue in many Latin American countries, receiving insufficient investment in research and development. Strategies for disease control and management currently lack efficient pharmaceuticals, commercial diagnostic kits with improved sensitivity, and vaccines. Genetic heterogeneity of Trypanosoma cruzi is a key aspect for novel drug design since pharmacological technologies rely on the degree of conservation of parasite target proteins. Therefore, there is a need to expand the knowledge regarding parasite genetics which, if fulfilled, could leverage Chagas disease research and development, and improve disease control strategies. The growing capacity of whole-genome sequencing technology and its adoption as disease surveillance routine may be key for solving this long-lasting problem.

Molecular epidemiology of Mycobacterium tuberculosis in Brazil before the whole genome sequencing era: a literature review.

Molecular-typing can help in unraveling epidemiological scenarios and improvement for disease control strategies. A literature review of Mycobacterium tuberculosis transmission in Brazil through genotyping on 56 studies published from 1996-2019 was performed. The clustering rate for mycobacterial interspersed repetitive units - variable tandem repeats (MIRU-VNTR) of 1,613 isolates were: 73%, 33% and 28% based on 12, 15 and 24-loci, respectively; while for RFLP-IS6110 were: 84% among prison population in Rio de Janeiro, 69% among multidrug-resistant isolates in Rio Grande do Sul, and 56.2% in general population in São Paulo. These findings could improve tuberculosis (TB) surveillance and set up a solid basis to build a database of Mycobacterium genomes.

Extensive genetic diversity of Plasmodium vivax dbp-II in Rio de Janeiro Atlantic Forest and Brazilian Amazon Basin: evidence of positive selection.

BACKGROUND: Plasmodium vivax is the most widespread human malaria parasite outside Africa and is the predominant parasite in the Americas. Increasing reports of P. vivax disease severity, together with the emergence of drug-resistant strains, underscore the urgency of the development of vaccines against P. vivax. Polymorphisms on DBP-II-gene could act as an immune evasion mechanism and, consequently, limited the vaccine efficacy. This study aimed to investigate the pvdbp-II genetic diversity in two Brazilian regions with different epidemiological patterns: the unstable transmission area in the Atlantic Forest (AF) of Rio de Janeiro and; the fixed malaria-endemic area in Brazilian Amazon (BA). METHODS: 216 Brazilian P. vivax infected blood samples, diagnosed by microscopic examination and PCR, were investigated. The region flanking pvdbp-II was amplified by PCR and sequenced. Genetic polymorphisms of pvdbp-II were estimated based on the number of segregating sites and nucleotide and haplotype diversities; the degree of differentiation between-regions was evaluated applying Wright's statistics. Natural selection was calculated using the rate of nonsynonymous per synonymous substitutions with the Z-test, and the evolutionary distance was estimated based on the reconstructed tree. RESULTS: 79 samples from AF and 137 from BA were successfully sequenced. The analyses showed 28 polymorphic sites distributed in 21 codons, with only 5% of the samples Salvador 1 type. The highest rates of polymorphic sites were found in B- and T cell epitopes. Unexpectedly, the nucleotide diversity in pvdbp-II was higher in AF (0.01) than in BA (0.008). Among the 28 SNPs detected, 18 are shared between P. vivax isolates from AF and BA regions, but 8 SNPs were exclusively detected in AF-I322S, K371N, E385Q, E385T, K386T, K411N, I419L and I419R-and 2 (N375D and I419M) arose exclusively in BA. These findings could suggest the potential of these geographical clusters as population-specific-signatures that may be useful to track the origin of infections. The sample size should be increased in order to confirm this possibility. CONCLUSIONS: The results highlight that the pvdbp-II polymorphisms are positively selected by host's immune pressure. The characterization of pvdbp-II polymorphisms might be useful for designing effective DBP-II-based vaccines.

Genomic analysis of bifunctional Class C-Class D ß-lactamases in environmental bacteria.

ß-lactamases, which are found in several bacterial species and environments, are the main cause of resistance to ß-lactams in Gram-negative bacteria. In 2009, a protein (LRA-13) with two ß-lactamase domains (one class C domain and one class D domain) was experimentally characterised, and an extended action spectrum against ß-lactams consistent with two functional domains was found. Here, we present the results of searches in the non-redundant NCBI protein database that revealed the existence of a group of homologous bifunctional ß-lactamases in the genomes of environmental bacteria. These findings suggest that bifunctional ß-lactamases are widespread in nature; these findings also raise concern that bifunctional ß-lactamases may be transferred to bacteria of clinical importance through lateral gene transfer mechanisms.

Whole genome sequence of Mycobacterium kansasii isolates of the genotype 1 from Brazilian patients with pulmonary disease demonstrates considerable heterogeneity.

Mycobacterium kansasii is an opportunistic pathogen and one of the most commonly encountered species in individuals with lung disease. We here report the complete genome sequence of 12 clinical isolates of M. kansasii from patients with pulmonary disease in Brazil.

Phylogenomic and genomic analysis reveals unique and shared genetic signatures of Mycobacterium kansasii complex species.

Species belonging to the Mycobacterium kansasii complex (MKC) are frequently isolated from humans and the environment and can cause serious diseases. The most common MKC infections are caused by the species M. kansasii (sensu stricto), leading to tuberculosis-like disease. However, a broad spectrum of virulence, antimicrobial resistance and pathogenicity of these non-tuberculous mycobacteria (NTM) are observed across the MKC. Many genomic aspects of the MKC that relate to these broad phenotypes are not well elucidated. Here, we performed genomic analyses from a collection of 665 MKC strains, isolated from environmental, animal and human sources. We inferred the MKC pangenome, mobilome, resistome, virulome and defence systems and show that the MKC species harbours unique and shared genomic signatures. High frequency of presence of prophages and different types of defence systems were observed. We found that the M. kansasii species splits into four lineages, of which three are lowly represented and mainly in Brazil, while one lineage is dominant and globally spread. Moreover, we show that four sub-lineages of this most distributed M. kansasii lineage emerged during the twentieth century. Further analysis of the M. kansasii genomes revealed almost 300 regions of difference contributing to genomic diversity, as well as fixed mutations that may explain the M. kansasii's increased virulence and drug resistance.

Outbreak report of polymyxin-carbapenem-resistant Klebsiella pneumoniae causing untreatable infections evidenced by synergy tests and bacterial genomes.

Polymyxin-carbapenem-resistant Klebsiella pneumoniae (PCR-Kp) with pan (PDR)- or extensively drug-resistant phenotypes has been increasingly described worldwide. Here, we report a PCR-Kp outbreak causing untreatable infections descriptively correlated with bacterial genomes. Hospital-wide surveillance of PCR-Kp was initiated in December-2014, after the first detection of a K. pneumoniae phenotype initially classified as PDR, recovered from close spatiotemporal cases of a sentinel hospital in Rio de Janeiro. Whole-genome sequencing of clinical PCR-Kp was performed to investigate similarities and dissimilarities in phylogeny, resistance and virulence genes, plasmid structures and genetic polymorphisms. A target phenotypic profile was detected in 10% (12/117) of the tested K. pneumoniae complex bacteria recovered from patients (8.5%, 8/94) who had epidemiological links and were involved in intractable infections and death, with combined therapeutic drugs failing to meet synergy. Two resistant bacterial clades belong to the same transmission cluster (ST437) or might have different sources (ST11). The severity of infection was likely related to patients' comorbidities, lack of antimicrobial therapy and predicted bacterial genes related to high resistance, survival, and proliferation. This report contributes to the actual knowledge about the natural history of PCR-Kp infection, while reporting from a time when there were no licensed drugs in the world to treat some of these infections. More studies comparing clinical findings with bacterial genetic markers during clonal spread are needed.

Uncovering Pseudogenes and Intergenic Protein-coding Sequences in TriTryps' Genomes.

Trypanosomatids belong to a remarkable group of unicellular, parasitic organisms of the order Kinetoplastida, an early diverging branch of the phylogenetic tree of eukaryotes, exhibiting intriguing biological characteristics affecting gene expression (intronless polycistronic transcription, trans-splicing, and RNA editing), metabolism, surface molecules, and organelles (compartmentalization of glycolysis, variation of the surface molecules, and unique mitochondrial DNA), cell biology and life cycle (phagocytic vacuoles evasion and intricate patterns of cell morphogenesis). With numerous genomic-scale data of several trypanosomatids becoming available since 2005 (genomes, transcriptomes, and proteomes), the scientific community can further investigate the mechanisms underlying these unusual features and address other unexplored phenomena possibly revealing biological aspects of the early evolution of eukaryotes. One fundamental aspect comprises the processes and mechanisms involved in the acquisition and loss of genes throughout the evolutionary history of these primitive microorganisms. Here, we present a comprehensive in silico analysis of pseudogenes in three major representatives of this group: Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi. Pseudogenes, DNA segments originating from altered genes that lost their original function, are genomic relics that can offer an essential record of the evolutionary history of functional genes, as well as clues about the dynamics and evolution of hosting genomes. Scanning these genomes with functional proteins as proxies to reveal intergenic regions with protein-coding features, relying on a customized threshold to distinguish statistically and biologically significant sequence similarities, and reassembling remnant sequences from their debris, we found thousands of pseudogenes and hundreds of open reading frames, with particular characteristics in each trypanosomatid: mutation profile, number, content, density, codon bias, average size, single- or multi-copy gene origin, number and type of mutations, putative primitive function, and transcriptional activity. These features suggest a common process of pseudogene formation, different patterns of pseudogene evolution and extant biological functions, and/or distinct genome organization undertaken by those parasites during evolution, as well as different evolutionary and/or selective pressures acting on distinct lineages.

Bio-Strings: A Relational Database Data-Type for Dealing with Large Biosequences.

DNA sequencers output a large set of very long biological data strings that we should persist in databases rather than basic text file systems. Many different data models and database management systems (DBMS) may deal with both storage and efficiency issues regarding genomic datasets. Specifically, there is a need for handling strings with variable sizes while keeping their biological meaning. Relational database management systems (RDBMS) provide several data types that could be further explored for the genomics context. Besides, they enforce integrity, consistency, and enable good abstractions for more conventional data. We propose the relational text data type to represent and manipulate biological sequences and their derivatives. We present a logical schema for representing the core biological information, which may be inferred from a given biological conceptual data schema and the corresponding function manipulations. We implement and evaluate these stored functions into an actual RDBMS for both efficacy and efficiency. We show that it is possible to enforce basic and complex requirements for the genomic domain. We claim that the well-established relational text data type in RDBMS may appropriately handle the representation and persistency of biological sequences. We base our approach on the idea of domain-specific abstract data types that can store data with semantically defined functions while hiding those details from non-technical end-users.

Correction: Balancing selection and high genetic diversity of Plasmodium vivax circumsporozoite central region in parasites from Brazilian Amazon and Rio de Janeiro Atlantic Forest.

[This corrects the article DOI: 10.1371/journal.pone.0241426.].

ProteinWorldDB: querying radical pairwise alignments among protein sets from complete genomes.

MOTIVATION: Many analyses in modern biological research are based on comparisons between biological sequences, resulting in functional, evolutionary and structural inferences. When large numbers of sequences are compared, heuristics are often used resulting in a certain lack of accuracy. In order to improve and validate results of such comparisons, we have performed radical all-against-all comparisons of 4 million protein sequences belonging to the RefSeq database, using an implementation of the Smith-Waterman algorithm. This extremely intensive computational approach was made possible with the help of World Community Grid, through the Genome Comparison Project. The resulting database, ProteinWorldDB, which contains coordinates of pairwise protein alignments and their respective scores, is now made available. Users can download, compare and analyze the results, filtered by genomes, protein functions or clusters. ProteinWorldDB is integrated with annotations derived from Swiss-Prot, Pfam, KEGG, NCBI Taxonomy database and gene ontology. The database is a unique and valuable asset, representing a major effort to create a reliable and consistent dataset of cross-comparisons of the whole protein content encoded in hundreds of completely sequenced genomes using a rigorous dynamic programming approach. AVAILABILITY: The database can be accessed through http://proteinworlddb.org

Whole-Genome Sequences of Mycobacterium abscessus subsp. massiliense Isolates from Brazil.

The Mycobacterium abscessus complex comprises multidrug-resistant, opportunistic, and rapidly growing pathogens responsible for severe infections. Here, we report the genome composition of four Mycobacterium abscessus subsp. massiliense isolates from three sources: two from the lung of a cystic fibrosis patient, one from a mammary cyst, and one from a gutter system.

A Murine Model of Mycobacterium kansasii Infection Reproducing Necrotic Lung Pathology Reveals Considerable Heterogeneity in Virulence of Clinical Isolates.

Among non-tuberculous mycobacteria, Mycobacterium kansasii is one of the most pathogenic, able to cause pulmonary disease indistinguishable from tuberculosis in immunocompetent susceptible adults. The lack of animal models that reproduce human-like lung disease, associated with the necrotic lung pathology, impairs studies of M. kansasii virulence and pathogenicity. In this study, we examined the ability of the C57BL/6 mice, intratracheally infected with highly virulent M. kansasii strains, to produce a chronic infection and necrotic lung pathology. As a first approach, we evaluated ten M. kansasii strains isolated from Brazilian patients with pulmonary disease and the reference strain M. kansasii ATCC 12478 for virulence-associated features in macrophages infected in vitro; five of these strains differing in virulence were selected for in vivo analysis. Highly virulent isolates induced progressive lung disease in mice, forming large encapsulated caseous granulomas in later stages (120-150 days post-infection), while the low-virulent strain was cleared from the lungs by day 40. Two strains demonstrated increased virulence, causing premature death in the infected animals. These data demonstrate that C57BL/6 mice are an excellent candidate to investigate the virulence of M. kansasii isolates. We observed considerable heterogeneity in the virulence profile of these strains, in which the presence of highly virulent strains allowed us to establish a clinically relevant animal model. Comparing public genomic data between Brazilian isolates and isolates from other geographic regions worldwide demonstrated that at least some of the highly pathogenic strains isolated in Brazil display remarkable genomic similarities with the ATCC strain 12478 isolated in the United States 70 years ago (less than 100 SNPs of difference), as well as with some recent European clinical isolates. These data suggest that few pathogenic clones have been widely spread within M. kansasii population around the world.

Balancing selection and high genetic diversity of Plasmodium vivax circumsporozoite central region in parasites from Brazilian Amazon and Rio de Janeiro Atlantic Forest.

Circumsporozoite protein (CSP) is the primary pre-erythrocytic vaccine target in Plasmodium species. Knowledge about their genetic diversity can help predict vaccine efficacy and the spread of novel parasite variants. Thus, we investigated pvcsp gene polymorphisms in 219 isolates (136 from Brazilian Amazon [BA], 71 from Rio de Janeiro Atlantic Forest [AF], and 12 from non-Brazilian countries [NB]). Forty-eight polymorphic sites were detected, 46 in the central repeat region (CR), and two in the C-terminal region. Also, the CR presents InDels and a variable number of repeats. All samples correspond to the VK210 variant, and 24 VK210 subtypes based on CR. Nucleotide diversity (π = 0.0135) generated a significant number of haplotypes (168) with low genetic differentiation between the Brazilian regions (Fst = 0.208). The haplotype network revealed similar distances among the BA and AF regions. The linkage disequilibrium indicates that recombination does not seem to be acting in diversity, reinforcing natural selection's role in accelerating adaptive evolution. The high diversity (low Fst) and polymorphism frequencies could be indicators of balancing selection. Although malaria in BA and AF have distinct vector species and different host immune pressures, consistent genetic signature was found in two regions. The immunodominant B-cell epitope mapped in the CR varies from seven to 19 repeats. The CR T-cell epitope is conserved only in 39 samples. Concerning to C-terminal region, the Th2R epitope presented nonsynonymous SNP only in 6% of Brazilian samples, and the Th3R epitope remained conserved in all studied regions. We conclude that, although the uneven distribution of alleles may jeopardize the deployment of vaccines directed to a specific variable locus, a unique vaccine formulation could protect populations in all Brazilian regions.

Exploration of Plasmodium vivax merozoite surface proteins 1 and 7 genetic diversity in Brazilian Amazon and Rio de Janeiro Atlantic Forest.

Plasmodium vivax merozoite surface proteins (PvMSP) 1 and 7 are considered vaccine targets. Genetic diversity knowledge is crucial to assess their potential as immunogens and to provide insights about population structure in different epidemiological contexts. Here, we investigate the variability of pvmsp-142, pvmsp-7E, and pvmsp-7F genes in 227 samples from the Brazilian Amazon (BA) and Rio de Janeiro Atlantic Forest (AF). pvmsp-142 has 63 polymorphisms - 57 nonsynonymous - generating a nucleotide diversity of π = 0.009 in AF, and π = 0.018 in BA. In pvmsp-7E, 134 polymorphisms - 103 nonsynonymous - generate the nucleotide diversity of π = 0.027 in AF, and π = 0.042 in BA. The pvmsp-7F has only two SNPs - A610G and A1054T -, with nucleotide diversity of π = 0.0004 in AF, and π = 0.0007 in BA. The haplotype diversity of pvmsp-142, pvmsp-7E, and pvmsp-7F genes is 0.997, 1.00, and 0.649, respectively. None of the pvmsp-142 or pvmsp-7E sequences are identical to the Salvador 1 strain's sequence. Conversely, most of pvmsp-7F sequences (94/48%) are identical to Sal-1. We evaluated eight B-cell epitopes in pvmsp-7E, four of them showed higher nucleotide diversity compared to pvmsp-7E's epitopes. Positive selection was detected in pvmsp-142, pvmsp-7E central region, and pvmsp-7F with Tajima's D. In pvmsp-7E, the significant nucleotide and haplotype diversities with low genetic differentiation, could be indicative of balancing selection. The genetic differentiation of pvmsp-142 (0.315) and pvmsp-7F (0.354) genes between AF and BA regions is significant, which is not the case for pvmsp-7E (0.193). We conclude that pvmsp-142 and pvmsp-7E have great genetic diversity even in AF region, an enclosure area with deficient transmission levels of P. vivax zoonotic malaria. In both Brazilian regions, pvmsp-119, pvmsp-7E, and pvmsp-7F are conserved, most likely due to their roles in parasite survival, and could be considered potential targets for a "blood-stage vaccine".

Quantitative Proteomic Map of the Trypanosomatid Strigomonas culicis: The Biological Contribution of its Endosymbiotic Bacterium.

Strigomonas culicis is a kinetoplastid parasite of insects that maintains a mutualistic association with an intracellular symbiotic bacterium, which is highly integrated into the protist metabolism: it furnishes essential compounds and divides in synchrony with the eukaryotic nucleus. The protist, conversely, can be cured of the endosymbiont, producing an aposymbiotic cell line, which presents a diminished ability to colonize the insect host. This obligatory association can represent an intermediate step of the evolution towards the formation of an organelle, therefore representing an interesting model to understand the symbiogenesis theory. Here, we used shotgun proteomics to compare the S. culicis endosymbiont-containing and aposymbiotic strains, revealing a total of 11,305 peptides, and up to 2,213 proteins (2,029 and 1,452 for wild type and aposymbiotic, respectively). Gene ontology associated to comparative analysis between both strains revealed that the biological processes most affected by the elimination of the symbiont were the amino acid synthesis, as well as protein synthesis and folding. This large-scale comparison of the protein expression in S. culicis marks a step forward in the comprehension of the role of endosymbiotic bacteria in monoxenous trypanosomatid biology, particularly because trypanosomatids expression is mostly post-transcriptionally regulated.

Systematic Identification and Classification of ß-Lactamases Based on Sequence Similarity Criteria: ß-Lactamase Annotation.

ß-lactamases, the enzymes responsible for resistance to ß-lactam antibiotics, are widespread among prokaryotic genera. However, current ß-lactamase classification schemes do not represent their present diversity. Here, we propose a workflow to identify and classify ß-lactamases. Initially, a set of curated sequences was used as a model for the construction of profiles Hidden Markov Models (HMM), specific for each ß-lactamase class. An extensive, nonredundant set of ß-lactamase sequences was constructed from 7 different resistance proteins databases to test the methodology. The profiles HMM were improved for their specificity and sensitivity and then applied to fully assembled genomes. Five hierarchical classification levels are described, and a new class of ß-lactamases with fused domains is proposed. Our profiles HMM provide a better annotation of ß-lactamases, with classes and subclasses defined by objective criteria such as sequence similarity. This classification offers a solid base to the elaboration of studies on the diversity, dispersion, prevalence, and evolution of the different classes and subclasses of this critical enzymatic activity.

Functional Analogy in Human Metabolism: Enzymes with Different Biological Roles or Functional Redundancy?

Since enzymes catalyze almost all chemical reactions that occur in living organisms, it is crucial that genes encoding such activities are correctly identified and functionally characterized. Several studies suggest that the fraction of enzymatic activities in which multiple events of independent origin have taken place during evolution is substantial. However, this topic is still poorly explored, and a comprehensive investigation of the occurrence, distribution, and implications of these events has not been done so far. Fundamental questions, such as how analogous enzymes originate, why so many events of independent origin have apparently occurred during evolution, and what are the reasons for the coexistence in the same organism of distinct enzymatic forms catalyzing the same reaction, remain unanswered. Also, several isofunctional enzymes are still not recognized as nonhomologous, even with substantial evidence indicating different evolutionary histories. In this work, we begin to investigate the biological significance of the cooccurrence of nonhomologous isofunctional enzymes in human metabolism, characterizing functional analogous enzymes identified in metabolic pathways annotated in the human genome. Our hypothesis is that the coexistence of multiple enzymatic forms might not be interpreted as functional redundancy. Instead, these enzymatic forms may be implicated in distinct (and probably relevant) biological roles.

Genomic surveillance: a potential shortcut for effective Chagas disease management

Chagas disease is an enduring public health issue in many Latin American countries, receiving insufficient investment in research and development. Strategies for disease control and management currently lack efficient pharmaceuticals, commercial diagnostic kits with improved sensitivity, and vaccines. Genetic heterogeneity of Trypanosoma cruzi is a key aspect for novel drug design since pharmacological technologies rely on the degree of conservation of parasite target proteins. Therefore, there is a need to expand the knowledge regarding parasite genetics which, if fulfilled, could leverage Chagas disease research and development, and improve disease control strategies. The growing capacity of whole-genome sequencing technology and its adoption as disease surveillance routine may be key for solving this long-lasting problem.

BioParser: a tool for processing of sequence similarity analysis reports.

UNLABELLED: The widely used programs BLAST (in this article, 'BLAST' includes both the National Center for Biotechnology Information [NCBI] BLAST and the Washington University version WU BLAST) and FASTA for similarity searches in nucleotide and protein databases usually result in copious output. However, when large query sets are used, human inspection rapidly becomes impractical. BioParser is a Perl program for parsing BLAST and FASTA reports. Making extensive use of the BioPerl toolkit, the program filters, stores and returns components of these reports in either ASCII or HTML format. BioParser is also capable of automatically feeding a local MySQL database with the parsed information, allowing subsequent filtering of hits and/or alignments with specific attributes. For this reason, BioParser is a valuable tool for large-scale similarity analyses by improving the access to the information present in BLAST or FASTA reports, facilitating extraction of useful information of large sets of sequence alignments, and allowing for easy handling and processing of the data. AVAILABILITY: BioParser is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 2.0 license terms (http://creativecommons.org/licenses/by-nc-nd/2.0/) and is available upon request. Additional information can be found at the BioParser website (http://www.dbbm.fiocruz.br/BioParser.html).